Project Summary In this proposal we will use Drosophila as a model system to understand how circadian (E24 hr) systems adapt to seasonal changes in environmental conditions, with an emphasis on the relatively uncharacterized role of temperature. A major foundation of this proposal is based on our long-standing work showing that in D. melanogaster the temperature-dependent splicing of the 3'-terminal intron (called dmpi8) from the critical clock component period (per) is a prominent 'thermosensor'that adjusts the distribution of daily wake-sleep cycles, eliciting seasonably appropriate responses. As in many diurnal animals, Drosophila manifests morning and evening activity bouts separated by a midday siesta and largely sleep during the night. On warm days splicing of the dmpi8 intron is inefficient leading to slow accumulation of per RNA levels, events that enhance midday siesta and likely minimize the risks associated with desiccation during the hot midday hours. Recent progress indicates that multiple suboptimal splicing signals [i.e., 5'and 3'splice sites (ss)] are the basis for the thermosensitivity in the splicing efficiency of dmpi8, and endow D. melanogaster with the ability to prolong its midday siesta into the mid-to-late afternoon, presumably facilitating its adaptation to temperate climates where warm days are typically associated with extended periods of heat. Indeed, temperature-dependent changes in per 3'-terminal splicing efficiency and adjustments in daily wake-activity cycles are absent in several species of Drosophila that are naturally restricted to Afro-equatorial localities, wherein temperature and daylength undergo little fluctuation throughout the year. Intriguingly, these non-thermal responsive species have strong 5'and 3'ss on their per 3'-terminal introns, perhaps limiting their ability to colonize temperate climates. Presumably, low temperatures favor the interaction of splicing factors with suboptimal splicing signals. We will undertake a multi-faceted experimental strategy that includes biochemical, molecular, cell-culture and whole animal approaches to understand the cis- and trans-acting factors regulating the splicing efficiency of per 3'-terminal introns and how they modulate wake-sleep cycles in Drosophila. This will include characterization of newly identified natural polymorphisms in per that differentially regulate dmpi8 splicing and might vary geographically. We will also determine whether the effects of dmpi8 splicing are preferentially mediated from the morning or evening rhythmic brain centers and/or the more recently described arousal/sleep pacemakers. By undertaking comparative studies using a wide variety of natural populations and Drosophila species, this proposed work offers a unique opportunity to integrate studies on gene expression and neural circuits controlling complex behaviors with ecological and evolutionary implications. On a broader perspective, our work suggests that natural selection operating at the level of splicing signals plays an important role in the thermal adaptation of life forms.